Enzymatic wound debriding compositions with enhanced enzymatic activity

ABSTRACT

The present invention is directed to topical enzymatic wound debriding compositions with enhanced enzymatic activity. These compositions comprise a dispersed phase comprising at least one proteolytic enzyme and at least one hydrophilic polyol; and a continuous phase comprising a hydrophobic base.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/267,730, filed 8 Dec. 2009. The contents of the referencedapplication are incorporated by reference.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to topical enzymatic wounddebriding compositions and methods of treating wounds in need ofdebridement.

B. Background

The healing of wounds is a complex process which is often furthercomplicated by the presence of non-viable, necrotic tissue in the woundarea. Debridement is the process of removing the non-viable tissue froma wound to prevent or diminish infection and facilitate healing. Topicalcompositions containing proteolytic enzymes such as trypsin, papain,bromelain, subtilisin, sutilains, and collagenase have been used forenzymatic wound debridement. Generally, the standard of care is to applythe composition to the wound in need of debridement once daily (onceevery 24 hours) or more often if the composition becomes soiled. Becausemany proteolytic enzymes are susceptible to degradation in water-basedcompositions, many wound debriding compositions are made with anhydrous,hydrophobic bases such as petrolatum, mineral oil and/or vegetable oilas disclosed in U.S. Pat. No. 3,821,364 and U.S. Pat. No. 6,479,060,both of which are herein incorporated by reference. However, enzymaticwound debriding compositions based on hydrophobic bases are generallynot miscible in the aqueous environment of a wound bed, and thus contactof the proteolytic enzyme with the wound bed is generally hindered. Someother compositions are made with anhydrous, hydrophilic bases such aspropylene glycol or poloxamers as disclosed in U.S. Pat. No. 6,548,556,US 2003/0198631 and US 2003/0198632, all of which are hereinincorporated by reference.

SUMMARY OF THE INVENTION

The present invention is directed to topical enzymatic wound debridingcompositions with enhanced enzymatic activity. These compositionscomprise a dispersed phase comprising at least one proteolytic enzymeand at least one hydrophilic polyol; and a continuous phase comprising ahydrophobic base. The wound debriding compositions of the presentinvention possess enhanced enzymatic activity over wound debridingcompositions of the prior art.

In one aspect of the present invention, there is disclosed a wounddebriding composition comprising a dispersed phase comprising a liquidhydrophilic polyol and at least one proteolytic enzyme; and a continuousphase comprising a hydrophobic base; wherein the amount of liquidhydrophilic polyol is within ±10% w/w of the optimum amount of theliquid hydrophilic polyol. For example, if the optimum amount was about30% w/w, the amount of liquid hydrophilic polyol that could be usedwould be between about 20% w/w and about 40% w/w of the totalformulation to achieve enhanced enzymatic activity of the formulation.In another aspect, the amount of liquid hydrophilic polyol is within±9%, 8%, 7%, or 6% w/w of the optimum amount of the liquid hydrophilicpolyol. In still another aspect, the amount of liquid hydrophilic polyolis within ±5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the optimum amountof the liquid hydrophilic polyol.

The “optimum amount of liquid hydrophilic polyol” in a compositioncomprising (a) a dispersed phase including a liquid hydrophilic polyoland at least one proteolytic enzyme; and (b) a continuous phasecomprising a hydrophobic base can be determined by the method describedin Section A of the Detailed Description section of this specification,which is incorporated into this section by reference.

A method for determining whether a composition is within +10% w/w of theoptimum amount of a liquid hydrophilic polyol is described in Section Bof the Detailed Description section of this specification, which isincorporated by reference.

The optimum amount of liquid hydrophilic polyol for compositions withdifferent proteolytic enzymes can differ. Additionally, the optimumamount of liquid hydrophilic polyol for compositions with a specificproteolytic enzyme can differ depending on the ingredients of thecomposition. For example, the optimum amount of liquid hydrophilicpolyol in a collagenase composition containing PEG-400 and petrolatumcan be different from the optimum amount of liquid hydrophilic polyol ina collagenase composition containing PEG-600 and petrolatum, ordifferent from a collagenase composition containing poloxamer-124 andpetrolatum.

The term “hydrophilic polyol” means water-soluble, polar aliphaticalcohols with at least two hydroxyl groups and includes, but is notlimited to, polymeric polyols (e.g., polyethylene glycols andpoloxamers).

The term “liquid” in the context of describing “hydrophilic polyol”,“polyethylene glycol”, or “poloxamer” means that the material is in theliquid state at 25° C.

The term “solid” in the context of describing “hydrophilic polyol”,“polyethylene glycol”, or “poloxamer” means that the material is in thesolid state at 25° C.

In another aspect of the present invention, there is disclosed a methodof treating a wound in need of debridement comprising: applying to thewound a composition comprising a dispersed phase comprising a liquidhydrophilic polyol, and an effective debriding concentration of at leastone proteolytic enzyme; and a continuous phase comprising a hydrophobicbase; wherein the amount of liquid hydrophilic polyol is within ±10% w/wof the optimum amount. In another aspect, the amount of liquidhydrophilic polyol is within ±9%, 8%, 7%, or 6% w/w of the optimumamount. In still another aspect, the amount of liquid hydrophilic polyolis within ±5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the optimum amount.

In some embodiments, the proteolytic enzyme is a metalloprotease, acysteine protease, a serine protease, or an aspartic peptidase.Generally, the optimum amount of hydrophilic polyol for compositionscomprising a metalloprotease, a cysteine protease or a serine proteaseis from about 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39% w/w to about 40% w/w, or any range or numericalamount derivable therein. The optimum amount of hydrophilic polyol forcompositions comprising an aspartic peptidase is from about 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67% w/w to about 68% w/w or any range or numerical amountderivable therein. In one embodiment the metalloprotease is collagenase.In another embodiment the metalloprotease is collagenase and the optimumamount of the hydrophilic polyol is from about 10%, 11%, 12%, 13%, 14%,15%, 16% 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% w/w to about 40%w/w or any range or numerical amount derivable therein. In oneembodiment, the metalloprotease is thermolysin. In another embodiment,the metalloprotease is thermolysin and the optimum amount hydrophilicpolyol is from about 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38% w/w to about 39% w/w orany range or numerical amount derivable therein. In one embodiment, thecysteine protease is papain. In another embodiment the cysteine proteaseis papain and the optimum amount of the hydrophilic polyol is from about19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38% w/w to about 39% w/w or any range ornumerical amount derivable therein. In one embodiment, the serineprotease is trypsin. In another embodiment the serine protease istrypsin and the optimum amount of hydrophilic polyol is from about 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%,20%, 21%, 22%, 23% w/w to about 24% w/w or any range or numericalderivable therein. In one embodiment, the aspartic peptidase is pepsin.In another embodiment the aspartic peptidase is pepsin and the optimumamount of hydrophilic polyol is from about 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67% w/wto about 68% w/w or any range or numerical amount derivable therein. Insome embodiments, the proteolytic enzyme is suspended in the dispersedphase. In other embodiments the proteolytic enzyme is dissolved in thedispersed phase.

In some embodiments, the liquid hydrophilic polyol is a liquidpolyethylene glycol or a liquid poloxamer, or mixtures thereof.

In some embodiments of the present invention, the dispersed phase mayfurther comprise a solid hydrophilic polyol in order to help physicallystabilize the composition or reduce or prevent phase separation. In someembodiments, the solid hydrophilic polyol is a solid poloxamer, or asolid polyethylene glycol, or mixtures thereof.

In various embodiments of the present invention, the hydrophobic basecomprises petrolatum, mineral oil, or vegetable oil, or mixturesthereof. In one embodiment, the base comprises petrolatum. In anotherembodiment, the hydrophobic base comprises a vegetable oil. In stillanother embodiment, the hydrophobic base comprises mineral oil. In afurther embodiment, the hydrophobic base comprises petrolatum andmineral oil, petrolatum and vegetable oil, mineral oil and vegetableoil, or petrolatum, mineral oil, and vegetable oil.

In still another embodiment, the hydrophobic base comprises a vegetableoil, wherein the vegetable oil is castor oil.

In one embodiment, the composition is a semisolid. In anotherembodiment, the composition is a liquid. In other embodiments, thecomposition is impregnated on a pad, gauze, or sponge. In oneembodiment, the composition is sterile or anhydrous or both.

The composition can be packaged in any package appropriate fordispensing a wound debrider. The compositions can be packaged inmulti-use, single-dose, or metered dose packages. Non-limiting examplesinclude a tube, bottle, jar, pump container, pressurized container,bladder container, aerosol container, aerosol spray container,non-aerosol spray container, syringe, pouch, or sachet.

In another embodiment of the present invention there is disclosed amethod of determining the optimum amount of liquid hydrophilic polyol toadd to a target composition comprising a dispersed phase including aproteolytic enzyme and a continuous phase including a hydrophobic base,the method comprising: (1) obtaining a series of compositions comprisingthe dispersed phase and the continuous phase, wherein the dispersedphase further includes a liquid hydrophilic polyol, and wherein eachcomposition in the series of compositions include an identical amount ofproteolytic enzyme and a different amount of the liquid hydrophilicpolyol; (2) determining the enzymatic activity of each composition inthe series of compositions; (3) determining the highest point on a graphthat plots the enzymatic activity versus the amount of liquidhydrophilic polyol(s) included in each composition of the series ofcompositions, wherein the highest point on the graph correlates to theoptimum amount of liquid hydrophilic polyol to add to the targetcomposition. In one aspect, the enzymatic activity of the series ofcompositions can be determined by using the in-vitro artificial eschartesting model as described in this specification.

In a further aspect of the present invention there is disclosed a methodof increasing enzymatic activity in a target composition comprising adispersed phase including a proteolytic enzyme and a continuous phaseincluding a hydrophobic base, the method comprising: (1) obtaining aseries of compositions comprising the dispersed phase and the continuousphase, wherein the dispersed phase further includes a liquid hydrophilicpolyol, and wherein each composition in the series of compositionsincludes an identical amount of proteolytic enzyme and a differentamount of the liquid hydrophilic polyol; (2) determining the enzymaticactivity of each composition in the series of compositions; (3)determining the highest point on a graph that plots the enzymaticactivity versus the amount of liquid hydrophilic polyol(s) included ineach composition of the series of compositions, wherein the highestpoint on the graph correlates to an optimum amount of liquid hydrophilicpolyol to add to the target composition, and (4) adding ±10% w/w of theoptimum amount of liquid hydrophilic polyol to the target composition,thereby increasing the enzymatic activity in the target composition. Inone aspect, the enzymatic activity of the series of compositions can bedetermined by using the in-vitro artificial eschar testing model asdescribed in this specification.

The amount of polyol in the series of compositions can vary from eachcomposition randomly or by a selected amount. In one embodiment, theamount of polyol in each composition of the series of compositions canbe 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% by weight orvolume of the composition.

The term “anhydrous” means that the compositions contain less than about5% w/w, or less than about 3% w/w, or less than about 1% w/w, or lessthan about 0.5% w/w, or less than about 0.1% w/w in relation to thetotal composition, or 0%, of free or added water, not counting the waterof hydration, bound water, or typical moisture levels present in any ofthe raw ingredients of the compositions.

Unless otherwise specified, the percent values expressed herein areweight by weight and are in relation to the total composition.

The use of the word “a” or “an” when used in conjunction with the term“comprising” or “containing” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device obtainingthe value, the method being employed to determine the value, or thevariation that exists among the objects being evaluated.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The terms “treating,” “inhibiting,” “preventing, or “reducing” or anyvariation of these terms, when used in the claims and/or thespecification includes any measurable decrease or complete inhibition toachieve a desired result.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. With respect to the transitionalphase “consisting essentially of,” and in one non-limiting aspect, abasic and novel characteristic of the compositions and methods disclosedin this specification includes the composition's enhanced enzymaticactivity.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising collagenase andPEG-400, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-400 comprised in the series ofcompositions x-axis).

FIG. 2. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising collagenase andPEG-600, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-600 comprised in the series ofcompositions x-axis).

FIG. 3. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising collagenase andpoloxamer-124, dispersed in a hydrophobic phase comprising whitepetrolatum (y-axis) versus the percentage of the poloxamer-124 comprisedin the series of compositions x-axis).

FIG. 4. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising trypsin andPEG-400, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-400 comprised in the series ofcompositions x-axis).

FIG. 5. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising papain andPEG-400, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-400 comprised in the series ofcompositions x-axis).

FIG. 6. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising thermolysin andPEG-400, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-400 comprised in the series ofcompositions x-axis).

FIG. 7. Plot of the in-vitro collagenolysis activity (mg/ml) of a seriesof compositions comprising a dispersed phase comprising pepsin andPEG-400, dispersed in a hydrophobic phase comprising white petrolatum(y-axis) versus the percentage of the PEG-400 comprised in the series ofcompositions x-axis).

FIG. 8. Plot of the physical release of collagenase (mg) from of aseries of compositions comprising a dispersed phase comprisingcollagenase and PEG-400, dispersed in a hydrophobic phase comprisingwhite petrolatum (y-axis) versus the percentage of the PEG-400 comprisedin the series of compositions x-axis).

FIG. 9. Enzyme stability in PEG-in-white petrolatum dispersion comparedwith oil-in-water emulsion cream.

FIG. 10. Debridement efficacy in Eschar removal in pig burn wound.

DETAILED DESCRIPTION

One aspect of the present invention provides for topical enzymatic wounddebriding compositions with enhanced enzymatic activity. Thesecompositions comprise a dispersed phase comprising at least oneproteolytic enzyme and a hydrophilic polyol; and a continuous phasecomprising a hydrophobic base. In one aspect of the invention, thehydrophilic polyol is a liquid hydrophilic polyol.

It was found that the enzymatic activity (e.g., in vitro collagenolysis)of the compositions of the present invention, which are dispersions of ahydrophilic polyol and a proteolytic enzyme in a hydrophobic base, notonly was higher than the enzymatic activity of enzyme compositions basedsolely on a proteolytic enzyme and hydrophobic base combination (i.e.,no hydrophilic phase such as a hydrophilic polyol), but alsosurprisingly higher than those enzyme compositions based solely on aproteolytic enzyme and hydrophilic base combination (i.e., nohydrophobic phase such as petrolatum). Since enzymes are activated inthe presence of moisture, it would have been expected to see the highestenzymatic activity in compositions based solely on a proteolytic enzymeand hydrophilic base combination, where the base would be completelymiscible in moisture and conditions would be the most favorable forrelease and activation of the enzyme. However, the dispersioncomposition of hydrophilic and hydrophobic phases of the presentinvention had the highest enzymatic activity correlating to an optimumamount of the hydrophilic polyol which was more than 0% and less than100% of the hydrophilic polyol in the composition.

It was found, expectedly, that the physical enzyme release incompositions based solely on a hydrophilic vehicle was greater than therelease of the enzyme in compositions based solely on a hydrophobicvehicle, and also more than compositions of the present invention. Asseen in FIG. 8, the enzyme release profile generally increased with theincreasing percentage of hydrophilic polyol (PEG-400), with the highestrelease at 100% and the lowest release at 0%. However, surprisingly, theenzymatic activity was greater with the dispersion compositions of thepresent invention (see FIGS. 1-7). Thus the enzymatic activity profileof these dispersion compositions does not correlate with the physicalenzyme release profile as would be expected.

The compositions of the present invention are suitable for treatment ofa wound in need of debridement by applying to the wound a compositioncomprising a dispersed phase comprising a hydrophilic polyol, and aneffective debriding concentration of at least one proteolytic enzyme;and a continuous phase comprising a hydrophobic base; wherein the amountof hydrophilic polyol is within ±10% w/w of the optimum amount, or ±9%,8%, 7%, or 6% w/w of the optimum amount, or ±5%, 4%, 3%, 2%, 1%, 0.5%,or 0.1% w/w of the optimum amount of hydrophilic polyol.

These and other non-limiting aspects of the present invention arediscussed in further detail in the following sections.

A. Method for Determining the Optimum Amount of Liquid HydrophilicPolyol

The following protocol can be used to prepare a series of compositions(referred to as “Series of Compositions”) and to subsequently determinethe optimum amount of liquid hydrophilic polyol that can be used in adispersion of the present invention:

Eleven (11) compositions can be used to create the Series ofCompositions. Note that the amount (% w/w) of proteolytic enzyme in theseries of compositions is held constant. The following steps can be usedto prepare the eleven (11) compositions:

(i) Determine the ingredients (i.e., liquid hydrophilic polyol,proteolytic enzyme, and hydrophobic base) to be used in the Series ofCompositions and select the amount of proteolytic enzyme to be used. Byway of example, liquid hydrophilic polyol (e.g., PEG 400), proteolyticenzyme (e.g., collagenase at 1% w/w), and hydrophobic base (e.g., whitepetrolatum).

(ii) For composition one in the Series of Compositions, use 0% of theliquid hydrophilic polyol, use the selected amount of proteolyticenzyme, and q.s the batch with the hydrophobic base to 100%. Forexample, and referring to step (i) above, composition one of the Seriesof Compositions would have: 0% w/w PEG 400, 99% w/w of white petrolatum,and 1% w/w of collagenase.

(iii) For composition two in the Series of Compositions, use 10% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%. (Note thatit is permissible to use some solid hydrophilic polyol in the makeup ofthe liquid hydrophilic polyol as necessary to produce a physicallystable dispersion for compositions in the Series of Compositions).

(iv) For composition three in the Series of Compositions, use 20% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(v) For composition four in the Series of Compositions, use 30% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(vi) For composition five in the Series of Compositions, use 40% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(vii) For composition six in the Series of Compositions, use 50% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(viii) For composition seven in the Series of Compositions, use 60% w/wof the liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(ix) For composition eight in the Series of Compositions, use 70% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(x) For composition nine in the Series of Compositions, use 80% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(xi) For composition ten in the Series of Compositions, use 90% w/w ofthe liquid hydrophilic polyol, the same amount of the proteolyticenzyme, and q.s. the batch with the hydrophobic base to 100%.

(xii) For composition eleven in the Series of Compositions, use 0% ofthe hydrophobic base, the same amount of the proteolytic enzyme, andq.s. the batch with the hydrophilic polyol.

(xiii) determine the enzymatic activity of each of the elevencompositions in the Series of Compositions by using the in vitroartificial eschar testing model for the following sample collectiontimes: 6, 12, 18 and 24 hours, as described in Section H of the DetailedDescription section of this specification.

(ivx) plot a curve of the enzymatic activity of each composition versusthe correlating amount of liquid hydrophilic polyol(s) present in eachcomposition of the Series of Compositions cumulatively for each datacollection time. The highest point on the curve for the cumulative24-hour data collection time correlates to the optimum amount of liquidhydrophilic polyol that can be used in a dispersion.

Further, given that multiple ingredients can be included in the Seriesof Compositions (e.g., polyol(s) proteolytic enzyme(s), hydrophobicbase, and additional ingredients within the dispersed phase, and/oradditional ingredients within the continuous hydrophobic phase), theSeries of Compositions can be created by (1) varying the amount ofhydrophilic polyol as discussed above for each composition in theseries, (2) using the determined amount of proteolytic enzyme, and (3)q.s.-ing the batch to 100% with the amount of the additional ingredientsincluding the hydrophobic base; except for composition eleven, where thebatch would be q.s.-ed to 100% with the amount of the additionalingredients including the hydrophilic polyol.

B. Method for Determining Whether a Composition has +/−10% w/w of theOptimum Amount of Liquid Hydrophilic Polyol

It can be determined if a composition comprising (a) a dispersed phaseincluding a liquid hydrophilic polyol and at least one proteolyticenzyme; and (b) a continuous phase comprising a hydrophobic base(referred to as “Composition of Interest”) is within +10% of the OptimumAmount of liquid hydrophilic polyol by using the following protocol:

Step One: Obtain a Composition of Interest that includes: (i) adispersed phase including a liquid hydrophilic polyol(s) and aproteolytic enzyme and (ii) a continuous phase including a hydrophobicbase.

Step Two: Prepare a series of compositions (referred to as “Series ofCompositions”) based on the Composition of Interest. Note that theamount (% w/w) of proteolytic enzyme in the Series of Compositions isheld constant and is the same as the amount (% w/w) present in theComposition of Interest. The following steps can be used to prepare theSeries of Compositions:

(i) Determine the amount of all ingredients in the Composition ofInterest (% w/w).

(ii) Determine the total amount of the continuous phase in theComposition of Interest (% w/w). By way of example, if the Compositionof Interest includes 15% w/w liquid hydrophilic polyol (e.g., PEG 400),1% w/w proteolytic enzyme (e.g., collagenase), and 84% w/w hydrophobicbase (e.g., white petrolatum), then the Composition of Interest would be84% w/w continuous phase and 16% w/w dispersed phase.

Step Three: Prepare the Series of Compositions in a manner describedabove in Section A of this specification (e.g., this would includepreparing 11 compositions in a manner described in Section A of thisspecification).

Step Four: Determine the enzymatic activity of each of the elevencompositions in the Series of Compositions by using the in vitroartificial eschar testing model for each of the following samplecollection times: 6, 12, 18 and 24 hours as described in Section H ofthe Detailed Description section of this specification.

Step Five: Plot a curve of the enzymatic activity of each compositionversus the correlating amount of liquid hydrophilic polyol(s) present ineach composition of the Series of Compositions cumulatively for eachdata collection time. The highest point on the curve for the cumulative24-hour data collection time correlates to the optimum amount of liquidhydrophilic polyol for the Composition of Interest.

Step Six: Compare the amount of liquid hydrophilic polyol present withinthe Composition of Interest to determine whether it is within ±10% w/wof the optimum amount of liquid hydrophilic polyol for the Compositionof Interest.

C. Proteolytic Enzymes

Any proteolytic enzyme useful for wound debridement is suitable for thepresent invention. Proteolytic enzymes (proteases) break down protein byhydrolysis of the peptide bonds that link amino acids together in thepolypeptide chain of a protein. They are divided into four major groupson the basis of catalytic mechanism: serine proteases, cysteineproteases. metalloproteases, and aspartic proteases. Some proteases havebeen identified with other catalytic amino acids in the active site,such as threonine and glutamic acid; however, they do not form majorgroups.

1. Serine Proteases

Serine proteases depend upon the hydroxyl group of a serine residueacting as the nucleophile that attacks the peptide bond. The major clansfound in humans include the chymotrypsin-like, the subtilisin-like, thealpha/beta hydrolase, and signal peptidase clans. In evolutionaryhistory, serine proteases were originally digestive enzymes. In mammals,they evolved by gene duplication to serve functions in blood clotting,the immune system, and inflammation. These proteases have a broadsubstrate specificity and work in a wide pH range. Non-limiting examplesof serine proteases include trypsin, chymotrypsin, subtilisin,sutilains, plasmin, and elastases.

2. Cysteine Proteases

Peptidases in which the nucleophile that attach the scissile peptidebond in the sulfhydryl group of a cysteine residue are known as cysteineproteases. Cysteine proteases are commonly encountered in fruitsincluding papaya, pineapple, and kiwifruit. Cysteine proteases have abroad specificity and are widely used under physiological conditions. Inthis family, papain has been used extensively for wound debridement fora long time. Other cysteine proteases, such as bromelain and analain,have also been investigated for the applications in wound debridement.Other non-limiting examples of cysteine proteases include calpain,caspases, chymopapain, and clostripain.

3. Metalloproteases

Metalloproteases are among the proteases in which the nucleophilicattach on a peptide bond is mediated by a water molecule, while adivalent metal cation, usually zinc but sometimes cobalt, manganese,nickel or copper, activates the water molecule. The metal ions areextremely important for the activity. Any compounds that have potentialto interact with the metal ion, chelating or oxidation, will affect theenzymatic activity. Non-limiting examples of metalloproteases in thisfamily include thermolysin, collagenases, matrix metallo proteinases(MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin,and various bacterial derived neutral metalloproteases.

4. Aspartic Peptidases

Aspartic peptidases are so named because aspartic acid residues are theligands of the activated water molecule. In most enzymes in this family,a pair of aspartic residues act together to bind and activate thecatalytic water molecule. All or most aspartic peptidases areendopeptidases. Most aspartic peptidases have a broad specificity.However, the optimum pH of most aspartic peptidases is in the acidicrange. Non-limiting examples of aspartic peptidases are pepsin,chymosin, beta-secretase, plasmepsin, plant acid proteases andretroviral proteases.

5. Collagenase

A suitable proteolytic enzyme for wound debridement is themetalloprotease collagenase. The collagenase can be substantially pureor it may contain detectable levels of other proteases.

The potency assay of collagenase, and meaning of “collagenase units” asused herein, is based on the digestion of undenatured collagen from(bovine Achilles tendon) at pH 7.2 and 37° C. for 24 hours. The numberof peptide bonds cleaved is measured by reaction with ninhydrin. Aminogroups released by a trypsin digestion control are subtracted. One netcollagenase unit will solubilize ninhydrin reactive material equivalentto 1 nanomole of leucine equivalents per minute.

The amount (potency or concentration) of collagenase in the compositionsof the present invention is at an effective level to debride the wound.Generally, the potency of collagenase in the compositions can vary fromabout 1 to about 10,000 collagenase units per gram of product, based onthe activity of the collagenase used in the product. In variousembodiments, the potency, expressed as collagenase units per gram ofproduct, is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500to about 10000, or any range or numerical amount derivable therein.

The concentration of collagenase in the compositions generally can varyfrom about 0.001% w/w to about 8% w/w. In various embodiments, theconcentration, expressed as percentage weight by weight, is from about0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.0100.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.050, 0.055, 0.060, 0.065,0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.125, 0.150, 0.175,0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75,0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7 to about 8 or any range ornumerical amount derivable therein.

In one embodiment, the collagenase is derived from Clostridiumhistolyticum; however, in other embodiments the collagenase can bederived from other sources. Methods for producing a suitable collagenaseare disclosed in U.S. Pat. Nos. 3,705,083; 3,821,364; 5,422,261;5,332,503; 5,422,103; 5,514,370; 5,851,522; 5,718,897; and 6,146,626 allof which are herein incorporated by reference.

6. Trypsin

Another suitable proteolytic enzyme for wound debridement is the serineprotease trypsin. Typically, trypsin is derived from the pancreas ofhealthy bovine or porcine animals, or both. Trypsin can also be derivedfrom recombinant sources. The pharmaceutical grade (USP/NF) of trypsinis known as Crystallized Trypsin. It contains not less than 2500 USPTrypsin Units per mg, calculated on the dried basis, and not less than90.0% and not more than 110.0% of the labeled potency. The potency assayof trypsin as well as the definition of a USP Trypsin Unit are found inthe Crystallized Trypsin monograph of the USP 31 (Official Aug. 1, 2008)herein incorporated by reference.

The amount (potency or concentration) of trypsin in the compositions ofthe present invention is at an effective level to debride the wound.Generally, the potency of trypsin in the compositions can vary fromabout 90 to about 60,000 USP Trypsin Units per gram of product. Invarious embodiments the potency of trypsin, expressed as USP TrypsinUnits per gram of product, is from about 90, 100, 150, 200, 250, 300,320, 350, 375, 400, 500, 600, 675, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 10000, 20000, 30000, 40000, 50000 to about 60000, or anyrange or numerical amount derivable therein.

The concentration of trypsin in the compositions generally can vary fromabout 0.0025% w/w to about 1% w/w. In various embodiments, theconcentration of trypsin, expressed as percent weight by weight, is fromabout 0.0025, 0.0050, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040,0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090,0.095, 0.10, 0.15, 0.20 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 to about 1, or any range ornumerical amount derivable therein.

D. Hydrophilic Polyols

Hydrophilic polyols of the present invention are water-soluble, polaraliphatic alcohols with at least two hydroxyl groups, and includepolymeric polyols, e.g., polyethylene glycols and poloxamers. In oneaspect of the invention, the hydrophilic polyol in the dispersed phaseis a liquid hydrophilic polyol. In some embodiments, the liquidhydrophilic polyol is a liquid polyethylene glycol or a liquidpoloxamer, or mixtures thereof. Solid hydrophilic polyols such as solidpolyethylene glycols or solid poloxamers can also be added to thedispersed phase of the invention to help physically stabilize thedispersion. Other examples of liquid hydrophilic polyols include but arenot limited to propylene glycol, butylene glycol, pentylene glycol,hexylene glycol, glycerin, hexylene glycol, methoxy polyethylene glycol,propylene carbonate, and ethoxydiglycol, and these may also be added tothe dispersed phase.

1. Polyethylene Glycols

Polyethylene glycols are homo-polymers of ethylene glycol and waterrepresented by the formula:

H(OCH₂CH₂)_(n)OH

in which n represents the average number of oxyethylene groups.Polyethylene glycols can be either liquid or solid at 25° C. dependingon their molecular weights.

The following suitable non-limiting examples of liquid polyethyleneglycols are described using USP nomenclature: polyethylene glycol 200,polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol500, and polyethylene glycol 600.

The following suitable non-limiting examples of solid polyethyleneglycols are described using USP nomenclature: polyethylene glycol 700,polyethylene glycol 800, polyethylene glycol 900, polyethylene glycol1000, polyethylene glycol 1100, polyethylene glycol 1200, polyethyleneglycol 1300, polyethylene glycol 1400, polyethylene glycol 1450,polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol1700, polyethylene glycol 1800, polyethylene glycol 1900, polyethyleneglycol 2000, polyethylene glycol 2100, polyethylene glycol 2200,polyethylene glycol 2300, polyethylene glycol 2400, polyethylene glycol2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethyleneglycol 2800, polyethylene glycol 2900, polyethylene glycol 3000,polyethylene glycol 3250, polyethylene glycol 3350, polyethylene glycol3750, polyethylene glycol 4000, polyethylene glycol 4250, polyethyleneglycol 4500, polyethylene glycol 4750, polyethylene glycol 5000,polyethylene glycol 5500, polyethylene glycol 6000, polyethylene glycol6500, polyethylene glycol 7000, polyethylene glycol 7500, andpolyethylene glycol 8000.

The liquid and solid polyethylene glycols are available commerciallyfrom the DOW Chemical Company under the CARBOWAX™ tradename and from theBASF Corporation under LUTROL® E and PLURACARE® E tradenames. Bothpharmaceutical grade (USP/NF) and cosmetic grade polyethylene glycolsare suitable for the present invention.

2. Poloxamers

Poloxamers are synthetic block copolymers of ethylene oxide andpropylene oxide represented by the formula:

HO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(a)H

in which formula a and b represent the number of repeat units. Generallya is from 2 to 150 and b is from 15 to 70 depending on the particularpoloxamer. Poloxamers can be either liquid or solid at 25° C. dependingon their molecular weights.

The following suitable non-limiting examples of liquid poloxamers aredescribed using CTFA/INCI nomenclature: poloxamer 101, poloxamer 105,poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer182, poloxamer 183, poloxamer 184, poloxamer 212, poloxamer 231,poloxamer 282, poloxamer 331, poloxamer 401, and poloxamer 402.

The following suitable non-limiting examples of solid poloxamers aredescribed using CTFA/INCI nomenclature: poloxamer 108, poloxamer 188,poloxamer 217, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer338, poloxamer 407, poloxamer 185, poloxamer 215, poloxamer 234,poloxamer 235, poloxamer 284, poloxamer 333, poloxamer 334, poloxamer335, and poloxamer 403.

The liquid and solid poloxamers are available commercially from the BASFCorporation under the PLURONIC® and LUTROL® tradenames and from theUNIQEMA Corporation under the SYNPERONIC® trademark. Pharmaceuticalgrade (USP/NF) poloxamers are poloxamer 124, poloxamer 188, poloxamer237, poloxamer 338, and poloxamer 407. Both pharmaceutical grade andcosmetic grade poloxamers are suitable for the present invention.

E. Hydrophobic Bases

The hydrophobic bases of the present invention can comprise, but are notlimited to, plant, animal, paraffinic, and synthetic derived fats,butters, greases, waxes, solvents, and oils; mineral oils, vegetableoils, petrolatum, water insoluble organic esters and triglycerides,silicones, or fluorinated compounds; or mixtures thereof. In oneembodiment of the present invention the hydrophobic phase comprisespetrolatum.

Plant derived materials include, but are not limited to, arachis(peanut) oil, balsam Peru oil, carnauba wax, candellila wax, castor oil,hydrogenated castor oil, cocoa butter, coconut oil, corn oil, cottonseed oil, jojoba oil, macadamia seed oil, olive oil, orange oil, orangewax, palm kernel oil, rapeseed oil, safflower oil, sesame seed oil, sheabutter, soybean oil, sunflower seed oil, tea tree oil, vegetable oil,and hydrogenated vegetable oil.

Non-limiting examples of animal derived materials include beeswax, codliver oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene,and tallow.

Non-limiting examples of paraffinic materials include isoparaffin,microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite,petrolatum, and paraffin.

Suitable non-limiting examples of organic esters and triglyceridesinclude C12-15 alkyl benzoate, isopropyl myristate, isopropyl palmitate,medium chain triglycerides, trilaurin, and trihydroxystearin.

Non-limiting examples of silicones are dimethicone and cyclomethicone. Anon-limiting example of a fluorinated compound ispolytetrafluoroethylene (PTFE).

1. Petrolatum

Petrolatum is a purified mixture of semisolid hydrocarbons obtained frompetroleum and varies from dark amber to light yellow in color. Whitepetrolatum is wholly or nearly decolorized petrolatum and varies fromcream to snow white in color. Petrolatum and White Petrolatum can alsovary in melting point, viscosity, and consistency.

Various grades are available commercially from the PENRECO Corporationunder the tradenames: PENRECO®ULTIMA, PENRECO®SUPER, PENRECO®SNOW,PENRECO®REGENT, PENRECO®LILY, PENRECO®CREAM, PENRECO®ROYAL,PENRECO®BLOND, and PENRECO®AMBER. Various grades are also availablecommercially from the SONNEBORN Corporation under the tradenames: ALBA®,SUPER WHITE PROTOPET®, SUPER WHITE FONOLINE®, WHITE PROTOPET 1S®, WHITEPROTOPET 2L®, WHITE PROTOPET 3C®, WHITE FONOLINE®, PERFECTA®, YELLOWPROTOPET 2A®, YELLOW FONOLINE®, PROTOLINE®, SONOJELL #4®, SONOJELL #9®,MINERAL JELLY #10®, MINERAL JELLY #14®, MINERAL JELLY #17®, ANDCARNATION TROUGH GREASE®.

Petrolatum and White Petrolatum are available in cosmetic grade andpharmaceutical (USP/NF) grade and both are suitable for the presentinvention.

F. Topical Compositions

The topical compositions of the present invention are dispersionscomprising a hydrophilic dispersed phase in a hydrophobic continuousphase. The dispersed phase comprises a proteolytic enzyme and ahydrophilic polyol. In an aspect of the invention, the hydrophilicpolyol is a liquid hydrophilic polyol. In some embodiments, the liquidhydrophilic polyol is a liquid polyethylene glycol or a liquidpoloxamer, or mixtures thereof. The continuous phase comprises ahydrophobic base. The hydrophobic base can be petrolatum. Thecompositions are useful for treatment of wounds for wound debridement.

The compositions can be anhydrous as defined herein. The compositionscan be semisolid or liquid. The composition can be impregnated on a pad,gauze, or sponge. The compositions can also be sterile.

The compositions can include additional materials known in the art thatare suitable for topical compositions of this nature, e.g., absorbents,deodorizers, surfactants, solvents, rheology modifiers, film formers,stabilizers, emollients, moisturizers, preservatives, antimicrobials,antioxidants, chelating agents, fragrances, and colorants.

The compositions can also include additional pharmaceutical activeingredients known in the art that are suitable for topical compositionsof this nature, e.g., antimicrobial agents, wound healing agents,anesthetic agents, vulnerary agents, and haemostatic agents. Anon-limiting example of a vulnerary agent is balsam Peru.

The compositions can be packaged in any package suitable for dispensinga wound debrider. The compositions can be packaged in multi-use,single-dose, or metered dose packages. Non-limiting examples include atube, bottle, jar, pump container, pressurized container, bladdercontainer, aerosol container, aerosol spray container, non-aerosol spraycontainer, syringe, pouch, or sachet.

G. Manufacturing Process

The compositions of the present invention can be prepared by techniquesand methods known by one of ordinary skill in the art by dissolving orsuspending the proteolytic enzyme in part or all of the availablehydrophilic polyol. The resulting solution or suspension can be mixedwith a hydrophobic base to form a dispersion, wherein the hydrophobicbase becomes the continuous phase and the hydrophilic polyol/enzymephase becomes the dispersed phase. These compositions can be preparedusing processing equipment known by one of ordinary skill in the art,e.g., blenders, mixers, mills, homogenizers, dispersers, dissolvers,etc.

H. In Vitro Artificial Eschar Testing Model

Enhancement of the enzymatic activity of the compositions wasestablished by testing the compositions using an in vitro artificialeschar model as described below and in the publication “Study on thedebridement efficacy of formulated enzymatic wound debriding agents byin vitro assessment using artificial wound eschar and by an in vivo pigmodel”, Shi et. al., Wound Repair Regen, 2009, 17(6):853, hereinincorporated by reference. Bovine collagen (Type I), bovine fibrinogen,and elastin were used to make an Artificial Wound Eschar (AWE)substrate. Collagen-FITC labeled, elastin-rhodamine, and fibrin-coumarinwere the raw materials used for producing the AWE substrate. To prepare1 gram of AWE substrate, 650 mg Collagen-FITC and 100 mg each ofelastin-rhodamine and fibrin-coumarin were weighed into a 50 mL tube andhomogenized in 10 mL of Tris buffer saline. In a separate tube, 10 mL offibrinogen solution was prepared at 15 mg/mL with Tris buffer saline.The two solutions were combined and thoroughly mixed. A thrombinsolution (0.25 mL at 50 U/mL) was added, quickly mixed, and the solutionwas poured into a Petri dish containing a 90 mm nonreactive membranefilter. As a result of the thrombin-induced fibrinogen polymerization,the material began to form a soft sheet on top of the membrane filter byclotting the dyed proteins into a solid matrix. The clotted AWEsubstrate was allowed to solidify for 30 minutes and then rinsed withwater for 15 minutes to remove the thrombin. The AWE substrate wasfurther dehydrated to 75% moisture content in preparation for use.

With the AWE substrate still attached to the membrane, a 35 mm diameterpiece was punched out using a hole punch. The AWE substrate punch wasplaced on the top flat face of a Franz Diffusion Cell System (HansonResearch, Chatsworth, Calif.), and a TEFLON® sample holder placed ontop. The debriding ointment samples were loaded in the center of thesample holder, and any excess sample was removed by scraping. Thesolution in the receptor cells was Tris buffer at a pH of 7.4 forsamples containing collagenase, papain, thermolysin, or trypsin; and wassodium acetate buffer at a pH of 2 for samples containing pepsin. Thesolution in receptor cells was sampled in 1 mL increments at thefollowing sample collection times: 0, 1, 2, 3, 6, 12, 18 and 24 hours.Once finished, the samples were analyzed by fluorescence measurement ofFITC dye at 485 nm (excitation wavelength) and 520 nm (emissionwavelength) to determine the digestion of collagen (collagenolysis)reported in mg/ml.

I. In-Vitro Physical Enzyme Release Test

The release of enzyme from the compositions was determined by a Franzcell diffusion study using PVDF (0.45 micron) filters. This study wasperformed at 35° C. and lasted for 6 hours. The solution samples in thereceptor cells were subjected to a total protein analysis.

The protein concentration was determined by a BCA assay (Peirce) whilethe same collagenase was used as the reference standard. The details aredescribed as follows.

The BCA Protein Assay combines the well-known reduction of Cu²⁺ to Cu¹⁺by protein in an alkaline medium with the highly sensitive and selectivecolorimetric detection of the cuprous cation (Cu¹⁺) by bicinchoninicacid. The first step is the chelation of copper with protein in analkaline environment to form a blue-colored complex. In this reaction,known as the biuret reaction, peptides containing three or more aminoacid residues form a colored chelate complex with cupric ions in analkaline environment containing sodium potassium tartrate. This becameknown as the biuret reaction because a similar complex forms with theorganic compound biuret (NH₂—CO—NH—CO—NH₂) and the cupric ion. Biuret, aproduct of excess urea and heat, reacts with copper to form a light bluetetradentate complex. In the second step of the color developmentreaction, BCA, a highly sensitive and selective colorimetric detectionreagent reacts with the cuprous cation (Cu¹⁺) that was formed in step 1.The purple-colored reaction product is formed by the chelation of twomolecules of BCA with one cuprous ion. The BCA/copper complex iswater-soluble and exhibits a strong linear absorbance at 562 nm withincreasing protein concentrations. The purple color may be measured atany wavelength between 550 nm and 570 nm with minimal (less than 10%)loss of signal. See the following reference herein incorporated byreference: Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson,B. J. and Klenk, D. C. (1985). Measurement of protein usingbicinchoninic acid. Anal. Biochem. 150, 76-85.

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the applicants to function well inthe practice of the invention. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Dispersions of Collagenase/PEG 400 in Petrolatum

The dispersions in TABLE 1 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 1 Wht Poloxamer Disper- PEG 400 PEG-1450 Petrolatum 407Collagenase sion % w/w % w/w % w/w % w/w % w/w A 0 0 99.8 0 0.2 B 10 089.8 0 0.2 C 15 0 84.8 0 0.2 D 20 0 79.9 0.4 0.2 E 30 0 69 0.9 0.2 F 500 49.3 1.2 0.2 G 68 0 29.8 2.0 0.2 H* 83 12.5 4.5 0 0.2 I* 70 29.8 0 00.2 *PEG-1450 was added to PEG-400 to form a semi-solid resulting inapproximate total PEG of 96% and 100% respectively.

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above and the resultsplotted in FIG. 1. As can be seen by the results in FIG. 1, the optimumamount of PEG-400 based on the 24 hour curve is about 20% w/w PEG-400.

Example 2 Dispersions of Collagenase/PEG 600 in Petrolatum

The dispersions in TABLE 2 were prepared with varying concentrations ofPolyethylene Glycol 600 (PEG-600) dispersed in Petrolatum.

TABLE 2 PEG 600 Wht Petrolatum Poloxamer 407 Collagenase Dispersion %w/w % w/w % w/w % w/w J 0 99.8 0 0.2 K 10 89.525 0.275 0.2 L 20 79.2480.552 0.2 M 30 68.973 0.827 0.2 N 50 48.42 1.38 0.2 O 80 17.59 2.21 0.2P 97 0 2.8 0.2

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 2. As can be seen by the results in FIG. 2, the optimumamount of PEG-600 based on the 24 hour curve is about 30% w/w PEG-600.

Example 3 Dispersions of Collagenase/Poloxamer 124 in Petrolatum

The dispersions in TABLE 3 were prepared with varying concentrations ofPoloxamer 124 dispersed in Petrolatum.

TABLE 3 Disper- Poloxamer 124 Wht Petrolatum Poloxamer 407 Collagenasesion % w/w % w/w % w/w % w/w Q  0 99.8 0 0.2 R 10 89.8 0 0.2 S 20 79.8 00.2 T 30 69.8 0 0.2 U 50 48.14 1.66 0.2 V 80 17.14 2.66 0.2 W  85* 0 150.2 *Poloxamer 407 was added to Poloxamer 124 to form a semi-solidresulting in approximate total of Poloxamer of 100%

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 3. As can be seen by the results in FIG. 3, the optimumamount of Poloxamer 124 based on the 24 hour curve is about 30% w/wPoloxamer 124.

Example 4 Dispersions of Trypsin/PEG 400 in Petrolatum

The dispersions in TABLE 4 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 4 Wht Disper- PEG 400 PEG 1450 Petrolatum Poloxamer 407 Trypsinsion % w/w % w/w % w/w % w/w % w/w X  0 0 99.8 0 0.2 Y 14 0 84.9 0.4 0.2Z 29 0 69.8 0.9 0.2 AA 59 0 39.16 1.64 0.2 BB 80 0 17.06 2.74 0.2 CC 82* 15.2 0 2.6 0.2 *PEG-1450 was added to PEG-400 to form a semi-solidresulting in approximate total PEG of 97%

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 4. As can be seen by the results in FIG. 4, the optimumamount of PEG-400 based on the 24 hour curve is about 14% w/w PEG-400.

Example 5 Dispersions of Papain/PEG 400 in Petrolatum

The dispersions in TABLE 5 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 5 Wht Disper- PEG 400 PEG 1450 Petrolatum Poloxamer 407 Papainsion % w/w % w/w % w/w % w/w % w/w DD  0 0 99.85 0 0.15 EE 15 0 85.050.4 0.15 FF 29 0 69.82 0.83 0.15 GG 43 0 54.85 1.24 0.15 HH 59 0 39.6941.636 0.15 II  82* 15.01 0 2.67 0.15 *PEG-1450 was added to PEG-400 toform a semi-solid resulting in approximate total PEG of 97%

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 5. As can be seen by the results in FIG. 5, the optimumamount of PEG-400 based on the 24 hour curve is about 29% w/w PEG-400.

Example 6 Dispersions of Thermolysin/PEG 400 in Petrolatum

The dispersions in TABLE 6 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 6 Wht Poloxamer Disper- PEG 400 PEG 1450 Petrolatum 407Thermolysin sion % w/w % w/w % w/w % w/w % w/w JJ  0 0 99.85 0 0.15 KK14 0 85.05 0.4 0.15 LL 29 0 69.82 0.83 0.15 MM 59 0 39.694 1.636 0.15 NN 82* 15.01 0 2.67 0.15 *PEG-1450 was added to PEG-400 to form asemi-solid resulting in approximate total PEG of 97%

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 6. As can be seen by the results in FIG. 6, the optimumamount of PEG-400 based on the 24 hour curve is about 29% w/w PEG-400.

Example 7 Dispersions of Pepsin/PEG 400 in Petrolatum

The dispersions in TABLE 7 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 7 Wht Disper- PEG 400 PEG 1450 Petrolatum Poloxamer 407 Pepsinsion % w/w % w/w % w/w % w/w % w/w OO  0 0 99 0 PP 15 0 84.2 0.4 1 QQ 290 68.97 0.83 1 RR 44 0 54.005 1.24 1 SS 58 0 38.844 1.636 1 TT  81*15.01 0 2.67 1 *PEG-1450 was added to PEG-400 to form a semi-solidresulting in approximate total PEG of 96%

The enzymatic debridement activity of each dispersion was determined bythe in-vitro artificial eschar model described above. The results areplotted in FIG. 7. As can be seen by the results in FIG. 7, the optimumamount of PEG-400 based on the 24 hour curve is about 58% w/w PEG-400.

Example 8 Dispersions of Collagenase/PEG 400 in Petrolatum for PhysicalRelease of Enzyme

The dispersions in TABLE 8 were prepared with varying concentrations ofPolyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.

TABLE 8 PEG 400 PEG 1450 Wht Petrolatum Collagenase Dispersion % w/w %w/w % w/w % w/w UU  0 0 99.8 0.2 VV  5 0 94.8 0.2 WW 10 0 89.8 0.2 XX 150 84.8 0.2 YY  83* 12.5 4.5 0.2 ZZ  70* 29.8 0 0.2 *PEG-1450 was addedto PEG-400 to form a semi-solid resulting in approximate total PEG of83% and 100% respectively.

The physical release of enzyme was determined by the In-vitro PhysicalEnzyme Release Test as described above. The results are plotted in FIG.8. As can be seen by the results in FIG. 8, the physical release ofcollagenase generally increased as the concentration of PEG-400 in thedispersion increased with the highest release at 100% and the lowestrelease at 0% PEG-400.

As can be seen by the results shown herein, the physical enzyme releaseprofile of the dispersions as a function of increased concentration ofhydrophilic polyol does not correlate to the enzymatic activity profileof the enzyme as a function of increased concentration of hydrophilicpolyol.

Example 9 Stability and Efficacy Data

FIG. 9 provides data comparing the stability of collagenase in adispersion of the present invention (“30% PEG in WP dispersion”) and anoil-in-water emulsion (“Aqueous cream”). These data suggest thatcollagenase was more stable in the 30% PEG in WP dispersion whencompared to the Aqueous cream. Tables 9-10 provide descriptions of the30% PEG in WP dispersion and Aqueous cream formulations.

TABLE 9 (30% PEG in WP dispersion)* Ingredients wt % PEG-600 30.059774Poloxamer-407 1.5078044 White Petrolatum 68.309516 Collagenase 0.1228163TOTAL 100 *PEG in WP dispersion was prepared as follows: (A) ActivePhase: (1) 9.71 grams of PEG-600 and 0.2361 grams of collagenase weremixed for 20 minutes at room temperature (20-25° C.) for 45 min. (B)Main Phase: (1) 102.784 grams of white petrolatum, 37.65 grams ofPEG-600, and 2.27 grams of poloxamer-407 were mixed at 70° C. untiluniform; (2) the mixture was cooled to 40-45° C. Added 7.79 grams of theActive Phase was added to the Main Phase followed by stirring for 30minutes or until homogenous mixture obtained.

TABLE 10 (Aqueous cream)* Ingredients wt % Isopropyl Myristate 30.57437Emulsifying Wax 4.502116 White Petrolatum 20.369574 Incroquat TMS4.502116 Water 20.009404 Glycerin (96%) 19.839324 Collagenase 0.2030955TOTAL 100 *Aqueous cream was prepared as follows: (A) Active Phase: (1)0.2 grams of collagenase was mixed with 20 grams of deionized water. (B)Main Phase: (1) 20.36 grams of white petrolatum was mixed with 4.5 gramsof emulsifying wax, 4.5 grams of Incroquate TMS, and 19.83 grams ofglycerin (96%) at 70° C. until uniform; (2) the mixture was cooled to35-40° C. Added Active Phase to Main Phase followed by stirring for 30minutes or until homogenous mixture obtained.

FIG. 10 provides data comparing enzyme debridement efficacy in escharremoval in pig burn wounds of a dispersion of the present invention(“PEG-in-White Petrolatum”—Table 11) to the following threeformulations: (1) an Aqueous cream—Table 12; (2) SANTYL® (“Commercialproduct”, which is a mixture of collagenase and white petrolatum); and ahydrogel formulation—Table 13. The burn wounds were created on pigs andhard eschars formed after several days. Formulation was applied to thehard eschars one a day for two weeks. Only fully debrided wounds werecounted as “complete debridement.” There were a total of 20 wounds pertreatment.

TABLE 11 (PEG-in-White Petrolatum)* Ingredients wt % Poloxamer-4070.99891551 White Petrolatum 78.7544989 Thermolysin 0.20168104 PEG-60020.0449046 TOTAL 100 *PEG-in-White Petrolatum was prepared as follows:(A) Active Phase: (1) 32.67 grams of PEG-600 and 1.63 grams ofPoloxamer-407 were homogenized at 70° C. until mixture was clear; (2)mixture was cooled to about 35° C.; and (3) thermolysin was and mixedfor at least 30 min.. (B) Main Phase: (1) 236.52 grams of whitepetrolatum, 30.05 grams of PEG-600, and 1.5 grams of poloxamer-407 werehomogenized at 70° C.; and (2) mixture was cooled to about 35° C. TheActive Phase (B) was added to the Main Phase (B) and mixed at roomtemperature (20-25° C.) for 45 min.

TABLE 12 (Aqueous Cream)* Ingredients wt % Emulsifying Wax 14.993927 1%KH2P04 in water (pH = 7.5) 74.057507 Isopropyl Palmitate, NF 5.4571649Glycerin 5.0104708 Thermolysin 0.2001065 Methyl paraben 0.2007937 Propylparaben 0.0800301 TOTAL 100 *Aqueous cream was prepared as follows: (1)parabens were melted in buffer at high temperature (>70° C.) along withglycerin; (2) emulsifying wax and isopropyl palmitate were added; (3)the mixture was mixed at high temperature for 45 min and then cooled toabout 35° C.; (4) thermolysin was added as a slurry in the buffer; (5)the mixture was cooled to room temperature (20-25° C.).

TABLE 13 (Hydrogel)* Ingredients wt % Hydroxypropylmethylcellulose2.250621745 1% KH2PO4 in water (pH = 7.5) 77.96851753 Thermolysin0.202530294 Methyl paraben 0.244719829 Propyl paraben 0.0480663Propylene glycol 19.28554438 TOTAL 100 *Hydrogel was prepared asfollows: (1) parabens and propylene glycol were solubilized in water at70° C.; (2) HPMC was added at room temperature (20-25° C.); (3)Thermolysin was added and a milky viscous solution formed.

1. A wound debriding composition comprising: (a) a dispersed phasecomprising a liquid hydrophilic polyol and at least one proteolyticenzyme; and (b) a continuous phase comprising a hydrophobic base;wherein the amount of the liquid hydrophilic polyol is within ±10% w/wof the optimum amount of the liquid hydrophilic polyol.
 2. Thecomposition of claim 1, wherein the amount of liquid hydrophilic polyolis within ±7% w/w of the optimum amount of the liquid hydrophilicpolyol.
 3. The composition of claim 1, wherein the amount of liquidhydrophilic polyol is within ±5% w/w of the optimum amount of the liquidhydrophilic polyol.
 4. The composition of claim 1, wherein theproteolytic enzyme is a metalloprotease.
 5. The composition of claim 4,wherein the metalloprotease is collagenase.
 6. The composition of claim4, wherein the metalloprotease is thermolysin.
 7. The composition ofclaim 1, wherein the proteolytic enzyme is a cysteine protease.
 8. Thecomposition of claim 7, wherein the cysteine protease is papain.
 9. Thecomposition of claim 1, wherein the proteolytic enzyme is a serineprotease.
 10. The composition of claim 9, wherein the serine protease istrypsin.
 11. The composition of claim 1, wherein the proteolytic enzymeis an aspartic peptidase.
 12. The composition of claim 11, wherein theaspartic peptidase is pepsin.
 13. The composition of claim 1, whereinthe liquid hydrophilic polyol is a liquid polyethylene glycol, or aliquid poloxamer, or mixtures thereof.
 14. The composition of claim 1,wherein the proteolytic enzyme is suspended in the dispersed phase. 15.The composition of claim 1, wherein the proteolytic enzyme is dissolvedin the dispersed phase.
 16. The composition of claim 1, wherein thehydrophobic base comprises petrolatum.
 17. The composition of claim 1,wherein the dispersed phase further comprises a solid hydrophilicpolyol.
 18. The composition of claim 17, wherein the solid hydrophilicpolyol is a solid polyethylene glycol, or a solid poloxamer, or mixturesthereof.
 19. The composition of claim 1, wherein the composition is asemisolid.
 20. The composition of claim 1, wherein the composition isanhydrous.
 21. The composition of claim 1, wherein the composition issterile.
 22. A method of treating a wound in need of debridementcomprising: applying to the wound a composition comprising a dispersedphase comprising a liquid hydrophilic polyol, and an effective debridingconcentration of at least one proteolytic enzyme; and a continuous phasecomprising a hydrophobic base; wherein the amount of liquid hydrophilicpolyol is within ±10% w/w of the optimum amount.
 23. (canceled)
 24. Amethod of determining an optimum amount of liquid hydrophilic polyol toadd to a target composition comprising a dispersed phase including aproteolytic enzyme and a continuous phase including a hydrophobic base,the method comprising: (1) obtaining a series of compositions comprisingthe dispersed phase and the continuous phase, wherein the dispersedphase further includes a liquid hydrophilic polyol, and wherein eachcomposition in the series of compositions include an identical amount ofproteolytic enzyme and a different amount of the liquid hydrophilicpolyol; (2) determining the enzymatic activity of each composition inthe series of compositions; (3) determining the highest point on a graphthat plots the enzymatic activity versus the amount of liquidhydrophilic polyol(s) included in each composition of the series ofcompositions, wherein the highest point on the graph correlates to theoptimum amount of liquid hydrophilic polyol to add to the targetcomposition. 25.-30. (canceled)
 31. A method of increasing enzymaticactivity in a target composition comprising a dispersed phase includinga proteolytic enzyme and a continuous phase including a hydrophobicbase, the method comprising: (1) obtaining a series of compositionscomprising the dispersed phase and the continuous phase, wherein thedispersed phase further includes a liquid hydrophilic polyol, andwherein each composition in the series of compositions includes anidentical amount of proteolytic enzyme and a different amount of theliquid hydrophilic polyol; (2) determining the enzymatic activity ofeach composition in the series of compositions; (3) determining thehighest point on a graph that plots the enzymatic activity versus theamount of liquid hydrophilic polyol(s) included in each composition ofthe series of compositions, wherein the highest point on the graphcorrelates to an optimum amount of liquid hydrophilic polyol to add tothe target composition, and (4) adding ±10% w/w of the optimum amount ofliquid hydrophilic polyol to the target composition, thereby increasingthe enzymatic activity in the target composition. 32.-36. (canceled)